Bacterial endotoxins, namely lipopolysaccharide (LPS), are common contaminants of many pharmaceutical, medical device, and food products that can elicit very strong immune responses in people and animals, sometimes resulting in septic shock. As such, monitoring for endotoxin in medical and food products is essential and a $6 billion annual market. Current methods for detecting endotoxin are complex, expensive, and difficult to standardize. Hence, there is an unmet need for rapid, simple assays that can be used to quickly assess endotoxin levels in field or point of care settings. Lateral flow based assays (LFAs) are ideally suited for end-users that require frequent, rapid, and straightforward endotoxin testing. LFAs require very little sample, no equipment or additional reagents, and are simple, robust and produced for $1-2 each at scale. We will develop a novel cleavage coupled competitive lateral flow assay (C3-LFA) that will enable inexpensive lateral flow devices that require no external instrumentation or skilled personnel to use. The basis of the assay technology is a novel peptide construct that incorporates the cleavage site for an endotoxin-activated protease between mimetope peptides that are specifically recognized by a particular monoclonal antibody. The intact dimer form of this construct has high affinity for the antibody but the monovalent affinity of the cleaved form is considerably weaker. Thus activity can unmask the antibody's antigen binding sites previously blocked by the dimer peptide construct by cleavage to low affinity monomers. The specific aims of this Phase I application are to 1) Optimize the dimer peptide and 2) Develop the LPS C3- LFA prototype. The long term goal of this project is to develop lateral flow immunoassays that can rapidly determine endotoxin levels above or below set thresholds. We have previously developed these mimetope peptides as lateral flow based sensors for their cognate antibody and are thus well poised to leverage that technique for the unique problem of enzyme activity via lateral flow.